Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) compete for nitrate in natural and engineered environments. A known important factor in this microbial competition is the ratio of available electron donor and elector acceptor, here expressed as Ac/N ratio (acetate/nitrate-nitrogen). We studied the impact of the Ac/N ratio on the nitrate reduction pathways in chemostat enrichment cultures, grown on acetate mineral medium. Stepwise, conditions were changed from nitrate limitation to nitrate excess in the system by applying a variable Ac/N ratio in the feed. We observed a clear correlation between Ac/N ratio and DNRA activity and the DNRA population in our reactor. The DNRA bacteria dominated under nitrate limiting conditions in the reactor and were outcompeted by denitrifiers under limitation of acetate. Interestingly, in a broad range of Ac/N ratios a dual limitation of acetate and nitrate occurred with co-occurrence of DNRA bacteria and denitrifiers. To explain these observations, the system was described using a kinetic model. The model illustrates that the Ac/N effect and concomitant broad dual limitation range related to the difference in stoichiometry between both processes, as well as the differences in electron donor and acceptor affinities. Population analysis showed that the presumed DRNA-performing bacteria were the same under nitrate limitation and under dual limiting conditions, whereas the presumed denitrifying population changed under single and dual limitation conditions.

Figure 1: Steady state reactor concentrations were measured for each cycle. Average deviations were obtained from daily measurements during the steady state (n > 3). (A) Ammonium concentrations measured and modeled for different Ac/N ratios. As a reference, the influent nitrate concentration is shown. (B) Biomass concentrations measured and modeled. The modeled fraction of DNRA biomass is shown for reference. (C) Nitrate and acetate concentrations, measured and modeled.

Mentions:
The initial culture was enriched and grown at a high Ac/N ratio of 1.87 mol/mol in the influent (Figure 1A). This resulted in nitrate limitation in the reactor, while acetate was in excess: 15 ± 2 % of the nitrate was assimilated and 70 ± 3% was reduced to ammonium via DNRA. The remaining 15% of nitrate was assumed to be reduced to dinitrogen gas, and thus denitrified. However, this remains to be verified. At this high Ac/N ratio, the biomass yields were 18.0 ± 1.1 g VSS/mole nitrate and 12.3 ± 1.6 g VSS/mole acetate (0.62 ± 0.04 mg protein/mg VSS) and the C/N content of the biomass was 0.22 ± 0.1 molN/molC. The redox potential in the reactor under these conditions was −450 mV and the color of the mixed culture was pink/reddish, due to high heme content of the biomass (redox spectra in Figure S1). These observations showed a good reproducibility of the previous enrichment in the same conditions (van den Berg et al., 2015).

Figure 1: Steady state reactor concentrations were measured for each cycle. Average deviations were obtained from daily measurements during the steady state (n > 3). (A) Ammonium concentrations measured and modeled for different Ac/N ratios. As a reference, the influent nitrate concentration is shown. (B) Biomass concentrations measured and modeled. The modeled fraction of DNRA biomass is shown for reference. (C) Nitrate and acetate concentrations, measured and modeled.

Mentions:
The initial culture was enriched and grown at a high Ac/N ratio of 1.87 mol/mol in the influent (Figure 1A). This resulted in nitrate limitation in the reactor, while acetate was in excess: 15 ± 2 % of the nitrate was assimilated and 70 ± 3% was reduced to ammonium via DNRA. The remaining 15% of nitrate was assumed to be reduced to dinitrogen gas, and thus denitrified. However, this remains to be verified. At this high Ac/N ratio, the biomass yields were 18.0 ± 1.1 g VSS/mole nitrate and 12.3 ± 1.6 g VSS/mole acetate (0.62 ± 0.04 mg protein/mg VSS) and the C/N content of the biomass was 0.22 ± 0.1 molN/molC. The redox potential in the reactor under these conditions was −450 mV and the color of the mixed culture was pink/reddish, due to high heme content of the biomass (redox spectra in Figure S1). These observations showed a good reproducibility of the previous enrichment in the same conditions (van den Berg et al., 2015).

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) compete for nitrate in natural and engineered environments. A known important factor in this microbial competition is the ratio of available electron donor and elector acceptor, here expressed as Ac/N ratio (acetate/nitrate-nitrogen). We studied the impact of the Ac/N ratio on the nitrate reduction pathways in chemostat enrichment cultures, grown on acetate mineral medium. Stepwise, conditions were changed from nitrate limitation to nitrate excess in the system by applying a variable Ac/N ratio in the feed. We observed a clear correlation between Ac/N ratio and DNRA activity and the DNRA population in our reactor. The DNRA bacteria dominated under nitrate limiting conditions in the reactor and were outcompeted by denitrifiers under limitation of acetate. Interestingly, in a broad range of Ac/N ratios a dual limitation of acetate and nitrate occurred with co-occurrence of DNRA bacteria and denitrifiers. To explain these observations, the system was described using a kinetic model. The model illustrates that the Ac/N effect and concomitant broad dual limitation range related to the difference in stoichiometry between both processes, as well as the differences in electron donor and acceptor affinities. Population analysis showed that the presumed DRNA-performing bacteria were the same under nitrate limitation and under dual limiting conditions, whereas the presumed denitrifying population changed under single and dual limitation conditions.